Biomarker identification and characterization holds great promise for more precise diagnoses and for tailored therapies. The heterogeneity of human cancers and unmet medical needs in these diseases provides a compelling argument to focus biomarker development in cancer. Mass spectrometry (MS)-based proteomics approaches have provided insight into biomarkers of cancer and other diseases with femtomole sensitivity and high analytical precision, but the results have proven difficult to reproduce owing to the complexity of human biofluids and to the protocols employed in these approaches. This CPTAC proposal will develop robust protocols and standards for MS proteomics employing both electrospray ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) platforms. Ion mobility MS provides several orders of magnitude increased dynamic range, and protocols for detection of cancer biomarker candidates will be further explored with this emerging technology platform. The consortium will employ high specificity immunologic reagents to develop platforms for precise detection and quantification of biomarkers of relevance for breast and prostate cancer. Immunoaffinity selection of specific biomarker proteins will be achieved as a sample fractionation step using nano-scale immunoaffinity columns prior to MS detection and quantification of specific proteins. An emerging technology incorporating specific antibodies on a microfabricated "bioCD" read by spinning disc interferometry enables label-free evaluation of hundreds of analytes from hundreds of samples in minutes. We will focus on candidate biomarkers relevant for breast and prostate cancer from the NF?B and STATS signaling pathways. Three for-profit and one not-for-profit corporate partners will join several academic groups with deep expertise and extensive resources in proteomics technologies to most efficiently evaluate and roll out robust protocols and standards for MS and affinity proteomics approaches. Prostate cancer samples will be made available from the NCI-sponsored ECOG trial;breast cancer patient and control samples will be collected specifically for this consortium by the Hoosier Oncology Group from throughout the State and region. Standardized sample collection organized from the Indiana University School of Medicine will ensure a diverse cross section of patient demographic groups. Three additional corporate partners will provide key technologies and consultation to improve team efficacy.